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Old 05-28-2012, 01:42 AM   #1
timforshew
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Question Primer design software for NGS amplicon sequencing of genes

Hi. I am looking into software packages to design primers for amplifying coding regions of genes. I intend to then sequence these on the Illumina MiSeq/HiSeq. I have been using the Fluidigm platform a lot and they have done a great job designing primers for me. I was wondering though if there are software packages where I can design the primers myself.

There are many packages out there (many based around primer3). The main thing I find hard though is designing primers that tile large exons well using amplicons of appropriate size. The best I have found is ExonPrimer (http://ihg.gsf.de/ihg/ExonPrimer.html). This worked very well for me for Sanger sequencing design but doesn’t seem to be that flexible for designing smaller amplicons needed for Illumina NGS.

Does anyone know of any other software that given a list of genes can design primers to produce amplicons (of a specific size range) to tile the coding regions?


Cheers
Tim
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Old 08-08-2012, 04:22 PM   #2
husamia
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Try optimus primer http://op.pgx.ca/
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Old 08-23-2012, 09:38 AM   #3
timforshew
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Thanks for the suggestion. I have given it a try but sadly with not much success. I need to design short amplicons so they can be sequenced on the Illumina platform. Also I am looking at degraded DNA so ideally want amplicons <200bp. I am not sure if I did something wrong but I tried designing primers to PTEN using this and if I try to use short amplicons it failed to design primers that tile the gene well. Any tips as to how best to get this software to work?

Thanks

Tim
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Old 08-23-2012, 11:44 AM   #4
NextGenSeq
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Why don't you just use DesignStudio on Illumina's website?
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Old 08-24-2012, 01:29 AM   #5
timforshew
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Thanks NextGenSeq. Unfortunately I need something to design standard PCR primers which I can use on the Fluidigm platform (as I understand it this just designs assays for Illumina's custom Enrichment and custom Amplicon systems?). I recently published work sequencing very small amounts of fragmented DNA with the Fluidigm platform and I am extending this with different genes.
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Old 03-27-2013, 09:18 AM   #6
Zaag
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Did optimus die? I can't reach the website for a couple of days now and using google... let's just say I learned a lot about autobots.
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Old 05-23-2013, 08:26 AM   #7
JeroenD
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Hi Tim,
Right now, I'm also designing primers for PTEN to use with the Fluidigm platform. The problem with the primer design is that different regions in exon 1 and 9 have in silico 2 PCR products, due to the pseudogene PTENP1. I couldn't find other primers because the similarity of these regions between PTEN and the pseudogene were high and large. Did you have the same problem? How did you solve this? I really need to exclude the pseudogene, but I'm also using the MiSeq so I can't have amplicons longer than 200 bp. Can you please give me some further information about this? It would be a major help for me.
Thanks!
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Old 05-24-2013, 06:34 AM   #8
timforshew
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Some regions are essentially impossible to look at specifically if you have short amplicons as there is so much similarity between PTEN and PTENP1. If you blat PTENP1 against PTEN you can spot the few locations they differ at then place the 3' end of your primers on them (although there still maybe be problems). Out of interest are you limited because of the MiSeq read length or because your DNA is degraded? We work with degraded DNA but if yours isnt you could use larger amplicons to "anchor" in specific regions and just tile more. Best wishes, Tim
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Old 05-28-2013, 02:28 AM   #9
JeroenD
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OK, thanks a lot! I am limited because of the MiSeq read length... Did you already find a good software to design primers? I'm working with Primer 3, but this is very time consuming. I'm looking for other softwares right now, I will let you know when I find a good one.
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Old 05-30-2013, 08:30 AM   #10
timforshew
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Unfortunately I havent found anything that is both good and free. Fluidigm do a good job designing primers. The ampliseq software also seems to do a pretty good job. Will update this is I find anything more. Tim
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Old 11-26-2013, 04:01 PM   #11
oncoapop
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We ported a version (beta and limited input - suggestions and comments welcomed) to the web for deep-sequencing validation experiments on our MiSeq.

http://www.myseqtools.com/
v0. takes SNV inputs only
v2. takes SNV inputs and nearby SNP/SNV/indels (annotated using non-standard characters or just N)

Workflow: hg18->hg19 (if applicable), designs primers based on primer3, performs in-silico PCR testing of primers, checks PE coverage on different MiSeq kits.

For those that pass criteria, it generates the primer order list (you can then add Illumina or Fluidigm adaptors yourself), an ampliconManifest for MiSeq and a html file for researchers to view amplicons & primers positions. It also outputs the failed positions so that you can design them with other tools / manually if necessary.

It is not polished yet as it was recently written and also by wet lab biologists using open sourced tools to support our existing pipeline running 2 MiSeq runs a week.

The web-server is slow so wait till the output is displayed (don't be impatient and close the browser or you will never get your results). If there is more interest, we will put it on a more powerful server.

If you are satisfied with the workflow but have more than 50 positions, I will need to run in on our in-house server (non-web based) and we can do this provided it is for academic (ie non-profit) research only and it is not like thousands of positions. Please fill in the form on the web.

Last edited by oncoapop; 11-26-2013 at 04:07 PM. Reason: Update reply
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